Julius Christina, Yuzenkova Yulia
Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK.
Nucleic Acids Res. 2017 Aug 21;45(14):8282-8290. doi: 10.1093/nar/gkx452.
Bacterial RNA polymerase is able to initiate transcription with adenosine-containing cofactor NAD+, which was proposed to result in a portion of cellular RNAs being 'capped' at the 5' end with NAD+, reminiscent of eukaryotic cap. Here we show that, apart from NAD+, another adenosine-containing cofactor FAD and highly abundant uridine-containing cell wall precursors, UDP-Glucose and UDP-N-acetylglucosamine are efficiently used to initiate transcription in vitro. We show that the affinity to NAD+ and UDP-containing factors during initiation is much lower than their cellular concentrations, and that initiation with them stimulates promoter escape. Efficiency of initiation with NAD+, but not with UDP-containing factors, is affected by amino acids of the Rifampicin-binding pocket, suggesting altered RNA capping in Rifampicin-resistant strains. However, relative affinity to NAD+ does not depend on the -1 base of the template strand, as was suggested earlier. We show that incorporation of mature cell wall precursor, UDP-MurNAc-pentapeptide, is inhibited by region 3.2 of σ subunit, possibly preventing targeting of RNA to the membrane. Overall, our in vitro results propose a wide repertoire of potential bacterial RNA capping molecules, and provide mechanistic insights into their incorporation.
细菌RNA聚合酶能够利用含腺苷的辅因子NAD⁺起始转录，有人提出这会导致一部分细胞RNA在5'端被NAD⁺“加帽”，这类似于真核生物的帽结构。在此我们表明，除了NAD⁺之外，另一种含腺苷的辅因子FAD以及高度丰富的含尿苷的细胞壁前体UDP-葡萄糖和UDP-N-乙酰葡糖胺在体外也能有效地用于起始转录。我们表明，起始过程中对NAD⁺和含UDP的因子的亲和力远低于它们在细胞内的浓度，并且用它们起始转录会刺激启动子逃逸。用NAD⁺起始转录的效率受利福平结合口袋氨基酸的影响，但含UDP的因子不受影响，这表明在利福平抗性菌株中RNA加帽发生了改变。然而，与NAD⁺的相对亲和力并不像之前所认为的那样取决于模板链的-1碱基。我们表明，成熟细胞壁前体UDP-胞壁酰五肽的掺入受到σ亚基3.2区域的抑制，这可能阻止了RNA靶向细胞膜。总体而言，我们的体外结果提出了一系列潜在的细菌RNA加帽分子，并为它们的掺入提供了机制上的见解。
